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Introduction

Background

The pharmaceutical and biotechnology industry’s goal is to discover therapeutic agents that are both safe and effective at treating or preventing diseases. Compounds identified as selective and potent in the early drug discovery phase are progressed to preclinical drug development for further evaluation. It is estimated that over 10% of drugs fail in clinical trials due to pharmacokinetic reasons (1), and the US Food and Drug Administration (FDA) guidelines emphasize the identification of metabolic pathways, relevant metabolites and potential drug-drug interactions for new chemical entities (NCEs) where metabolism is the principal route of elimination (2) Consequently, drug metabolism studies are a critical constituent of any drug development program.

The primary site of metabolism for many drugs is the liver. Liver-derived systems such as liver slices, sub-cellular liver fractions, and intact hepatocytes are typically utilized to assess the metabolism of NCEs. Intact hepatocytes contain the cytochrome P450’s (CYPs), other non-P450 enzymes, and phase II enzymes such as sulfo- and glucuronosyltransferases, and thus represent a prime model system for studying drug disposition in vitro (3). NCEs can be screened and rank-ordered according to metabolic half-life estimates or in vitro intrinsic clearance values (Clint,in vitro) obtained from metabolic stability studies. Moreover, metabolic screening assays enable drug developers to focus on the improvement of compounds through structural activity relationships (SAR) and prevent the progression of labile compounds to more costly in vivo studies. Given that cryopreserved hepatocytes retain enzymatic activities similar to those of fresh hepatocytes and offer convenience to the end user (4), the utility of cryopreserved hepatocytes in efforts to define a drug’s disposition in vitro is advantageous as compared to other model systems.

Important notes

Review this protocol to ensure y ou have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not maintain viability if refrozen.

Protocol

Advanced Preparation

Prepare Incubation Medium by combining Hepatocyte Maintenance Supplement Pack (Serum-free) with Williams Medium E per kit instructions, and warm to 37°C in a water bath. At least 5 mL of Incubation Medium will be needed per test article and control.

Prepare compound stocks: test articles (TA) and positive control(s) (PC) dissolved in an organic solvent such as methanol or DMSO to desired concentration, such as 1 mM.

In separate conical tubes, add the test compounds and positive control(s) to warm Incubation Medium to yield the desired working concentration(s). For example, prepare 2 μM by adding 10 μL of 1 mM test article stock solution to 5 mL of Incubation Medium. Note: if DMSO is used as a solvent, the concentration should not exceed 0.1%, with a maximum of 1% in the final Incubation Medium.

Pipette 0.5 mL of Incubation Medium containing the test article or positive control into respective wells of a 12-well non-coated plate. See Figure 1. Note: Final substrate concentration will be 1 μM once step 6 is complete.

Place the plate in the incubator on an orbital shaker to allow the substrates to warm for approximately 5-10 min prior to initiation of the reactions.

For the negative control, boil 1.0 x 106 viable hepatocytes/mL for 5 min to eliminate enzymatic activity. Use enough volume to cover the number of negative controls desired.

Remove the 12-well non-coated plate containing the substrates from the incubator.

Start reactions by adding 0.5 mL of 1.0 x 106 viable cells/mL in each appropriate well of the plate to yield a final cell density of 0.5 x 106 viable cells/mL. Pipette 0.5 mL of the inactivated hepatocytes into the negative control wells.

Return the plate to the orbital shaker in the incubator and adjust the shaker speed to 90-120 rpms.

Determine the in vitro half-life (t1/2) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.

Intrinsic clearance in vitro (Clint in vitro) can be calculated according to the equation: Clint in vitro = kV/N, where k = 0.693/t1/2, V = incubation volume (1 mL) and N = number of hepatocytes per well (0.5 x 106 viable cells).

Clint in vitro may be scaled to in vivo predictions according to Obach (5) and McGinnity (4).